Apolipoprotein B, the major apolipoprotein on atherogenic VLDL and LDL, is essential for the assembly and secretion of these lipoproteins by human livers. Studies in cultured liver cells have provided important insights related to the unique regulation of apoB, including its inefficient translocation, its bitopic topology across the endoplasmic reticulum (ER) membrane, its degradation by the cytosolic proteasome, and some of the characteristics of its association with its lipid ligands to become a lipoprotein. However, several critical aspects of the regulation of the assembly and secretion of apoB-lipoproteins remain undefined. In the proposed studies, we will continue to use cultured hepatocyte cell-lines, particularly HepG2 and McARH7777 cells, to address unanswered questions. We will, however, extend our investigations to include studies using primary mouse hepatocytes as well as in vivo measurements of apoB-lipoprotein secretion in adenovirus-infected, transgenic, and "knockout" mouse models. Four hypotheses will be tested: Hypothesis A: The complex secondary structure of apoB, and in particular the presence of one or more densely hydrophobic b-sheet domains, is the basis for the unique aspects of its translocation across the ER and its predisposition for ubiquitination and proteasomal degradation. Hypothesis B: Newly synthesized core lipids, triglycerides (TG) and cholesteryl esters (CE) are both critical for the efficient initial translocation of apoB across the ER and its targeting for lipoprotein assembly. However, TG is the sole lipid added at the later stages of lipoprotein assembly ("the second step"). Hypothesis C: The transition of apoB from a lipid-poor lipoprotein to a TG-enriched lipoprotein that is targeted for secretion includes the completion of translation and translocation, and transport of the lipid-poor particle to either the ER Golgi Intermediate Compartment (ERGIC) or the Golgi apparatus. It is at one of these two sites that bulk lipid addition (the second step) occurs. Importantly, this trafficking can be regulated by a variety of mechanisms distinct from core-lipid synthesis. Hypothesis D: Fatty acid flux to the liver is the major determinant of the assembly and secretion of apoB-lipoproteins. The impact of fatty acid flux on apoB-lipoprotein secretion may be modulated by the status of insulin signaling in the liver, but the latter plays only a minor role in the overall regulation of apoB-lipoprotein secretion. We believe that completion of these studies will provide the basis for new and innovative approaches to the treatment of human dyslipidemias.